Composition of a first non-labeled monoclonal antibody binding to a tumor antigen and a non-cross reactive second monoclonal antibody labeled with a NIR fluorescence label

ABSTRACT

This invention relates to a composition of a non-labeled monoclonal antibody binding to a tumor antigen and a second monoclonal antibody labeled with a NIR fluorescence label, binding to the same tumor antigen, wherein the first and second antibody exhibit no cross reactivity. The composition can be used for the treatment of patients suffering of solid tumors which are associated with an overexpression of such a tumor antigen. The invention further relates to a the co-administration of said first and second antibody as wells as to a method of acquiring a NIR fluorescence images of such tumors or the patients suffering from such tumors during the treatment of said patient with such composition.

This invention relates to a composition of a non-labeled monoclonalantibody binding to a tumor antigen and a second monoclonal antibodylabeled with a NIR fluorescence label, binding to the same tumorantigen, wherein the first and second antibody exhibit no crossreactivity. The composition can be used for the treatment of patientssuffering of solid tumors which are associated with an overexpression ofsuch a tumor antigen. The invention further relates to theco-administration of said first and second antibody as well as to amethod of acquiring a NIR fluorescence images of such tumors or thepatients suffering from such tumors during the treatment with suchcomposition or the co-administration of such antibodies.

BACKGROUND OF THE INVENTION

Monoclonal Antibodies in the Therapy

In an ongoing quest to improve the therapeutic arsenal against cancer, afourth weapon other than surgery, chemotherapy and radiotherapy hasemerged, i.e. targeted therapy. Targeted therapy includes tyrosinekinase receptor inhibitors (small molecule inhibitors like imatinib,gefitinib, and erlotinib), proteasome inhibitors (bortezomib),biological response modifiers (denileukin diftitox) and monoclonalantibodies (MAbs). The remarkable specificity of MAbs as targetedtherapy makes them promising agents for human therapy. Not only can MAbsbe used therapeutically to protect against disease, they can also beused to diagnose a variety of illnesses, measure serum protein and druglevels, type tissue and blood and identify infectious agents andspecific cells involved in immune response. About a quarter of allbiotech drugs in development are MAbs, and about 30 products are in useor being investigated. A majority of the MAbs are used for the treatmentof cancer. (Gupta, N.; et al., Indian Journal of Pharmacology 38 (2006)390-396; Funaro, A.; et al., Biotechnology Advances 18 (2000) 385-401;Suemitsu, N. et al., Immunology Frontier 9 (1999) 231-236)

Labeled Monoclonal Antibodies and In-Vivo Imaging

Several in vivo imaging methods are available for the quantification oftherapeutic antibodies in tumor tissue usually based on labeledderivatives of the antibodies. Said labeled antibodies usually includeantibodies labeled with radiolabels such as, e.g. ¹²⁴I, ¹¹¹In, ⁶⁴Cu, andothers, for use in positron emission tomography (PET) (see e.g.Robinson, M. K., et al., Cancer Res 65 (2005) 1471-1478; Lawrentschuk,N., et al., BJU International 97 (2006) 916-922; Olafsen, T., et al.,Cancer Research 65 (2005) 5907-5916; and Trotter, D. E., et al., Journalof Nuclear Medicine 45 (2004) 1237-1244), ¹²³I, ¹²⁵I, and ^(99m)Tc andothers for use in single photon emission computed tomography (SPECT)(see e.g. Orlova, A., et al., Journal of Nuclear Medicine 47 (2006)512-519; Dietlein, M., et al., European Journal of Haematology 74 (2005)348-352).

Also nonradioactive labels are known for in-vivo imaging techniques,e.g. near-infrared (NIR) fluorescence labels, activatable dyes, andengodogenous reporter groups (fluorescent proteins like GFP-likeproteins, and bioluminescent imaging) (Licha, K., et al., Adv Drug DelivRev, 57 (2005) 1087-1108). Especially NIR fluorescence imaging can beused for the quantification of therapeutic antibodies in tumor tissue.Advantages of near infrared imaging over other currently used clinicalimaging techniques include the following: potential for simultaneous useof multiple, distinguishable probes (important in molecular imaging);high temporal resolution (important in functional imaging); high spatialresolution (important in vivo microscopy); and safety (no ionizingradiation).

There exist different monoclonal antibodies covalently coupled to a NIRfluorescence label (Hilger, I., et al, Eur Radial (2004) 1124-1129;Ballou, B., et al., Cancer Immunol Immunother. 41 (4) (1995) 257-63;Ballou, B., et al., Proceedings of SPIE—The International Society forOptical Engineering 2680 (1996) 124-131; Ballou, B., et al., BiotechnolProg. (1997) 649-58; Ballou, B., et al., Cancer detection and prevention(1998), 22 251-257 Becker, A., et al., Nature Biotechnology 19 (2001)127-131; Montet, X., et al., Cancer Research 65 (2005), 6330-6336;Rosenthal, E. L., et al., The Laryngoscope 116 (2006) 1636-1641; EP1619501, WO 2006/072580, WO 2004/065491 and WO 2001/023005) which wereused as single agents for NIR fluorescence imaging.

In NIR fluorescence imaging, filtered light or a laser with a definedbandwidth is used as a source of excitation light. The excitation lighttravels through body tissues. When it encounters a near infraredfluorescent molecule (“contrast agent”), the excitation light isabsorbed.

The fluorescent molecule then emits light (fluorescence) spectrallydistinguishable (slightly longer wavelength) from the excitation light.Despite good penetration of biological tissues by near infrared light,conventional near infrared fluorescence probes are subject to many ofthe same limitations encountered with other contrast agents, includinglow target/background ratios.

Near infrared wavelengths (approximately 640-1300 nm) have been used inoptical imaging of internal tissues, because near infrared radiationexhibits tissue penetration of up to 6-8 centimeters. See, e.g., Wyatt,J. S., and Kirkpatrick, P. J., Phil. Trans. R. Soc. B 352 (1997)701-705; Tromberg, et al., Phil. Trans. R. Soc. London B 352 (1997)661-667.

The exact amounts of the antibody-label conjugates used for in vivoimaging depends on the different characteristics and aspects of thelabels used, e.g. for NIR fluorescence labels the quantum yield of thelabel is one of the criteria for the amount of label or labeled antibodyused (see e.g. WO 2006/072580).

Therapy Monitoring During Treatment with Monoclonal Antibodies

Factors affecting the successful therapy of malignant diseases includethe antibody dose used and the schedule of administration, the half-lifeand fast blood clearance of the antibodies, the presence of circulatingantigen, poor tumor penetration of the high/mol.-wt. monoclonal antibody(MAb) and the way in which these molecules are catabolized. At present,there is a lack of knowledge about many aspects of the physiologicalfunction and metabolism of antibodies. (Iznaga-Escobar, N. et al, Meth.Find. Exp. Clin. Pharm. 26(2) (2004) 123-127). Therefore it is importantto monitor the course of such therapies.

The success of such treatments is usually assessed using differentimaging techniques like chest X-ray, computed tomography (CT),computerized axial tomography (CAT), molecular resonance imaging (MRI),positron emission tomography (PET), single photon emission computedtomography (SPECT), fluorescence imaging (FI), and bioluminescentimaging (BLI) (see e.g. Helms, M. W, et al., Contributions tomicrobiology 13 (2006) 209-231 and Pantel, K., et al., JNCI 91 (1999)1113-1124). It is often defined as a “Response” to the treatment.According to RECIST criteria tumor response for solid tumors (Therasse,et al., J. Nat. Cancer Institute. 92 (2000) 205-216) is categorized independency of the volume progression or regression of the tumors (e.g.measured via CT) into four levels: complete response (CR) or partialresponse (PR), stable disease (SD) and progressive disease (PD) (seeTable 1). Furthermore the European Organization for Research andTreatment of Cancer (EORTC) proposed a categorization into four levelsin dependency of the metabolism of the tumors measured via2-[¹⁸F]-Fluoro-2-deoxyglucose positron emission tomography (FDG-PET)(Young, H., et al., Eur J Cane 35 (1999) 1773-1782 and Kelloff, G. J.,et al, Clin Cane Res 11 (2005) 2785-2808): complete metabolic response(CMR) or partial metabolic response (PMR), stable metabolic disease(SMD) and progressive metabolic disease (PMD) (see Table 2). Recently acombined assessment with CT and PET gets more and more common. While CTmainly focuses on the development of tumor size it delivers onlyrestricted information on the tumor metabolism and is associated withexposure to radioactive radiation, PET imaging gives more insight in thetumor metabolism, but still radioactive labels are needed for thistechnique.

SUMMARY OF THE INVENTION

The invention comprises a pharmaceutical composition comprising

-   -   a) a non-labeled monoclonal antibody binding to a tumor antigen        and    -   b) a second monoclonal antibody labeled with a NIR fluorescence        label, specifically binding to the same tumor antigen,    -   characterized in that, the first and second antibody exhibit no        cross reactivity.

Such composition can be composed of either one compartment comprisingboth antibodies or of two compartments, one comprising the non-labeledmonoclonal antibody and one comprising the labeled monoclonal antibody

Preferably the non-labeled monoclonal antibody is an anti-HER2 antibody,preferably trastuzumab or pertuzumab.

In another preferred embodiment the non-labeled monoclonal antibody isan anti-EGFR antibody, preferably caetuximab or rhMab ICR62.

One embodiment of the invention is the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a patient suffering from a solid tumoroverexpressing said tumor antigen

wherein the non-labeled monoclonal antibody is co-administered with asecond monoclonal antibody labeled with a NIR fluorescence label,specifically binding to the same tumor antigen, characterized in thatthe first and second antibody exhibit no cross reactivity.

Another embodiment of the invention is the use of a non-labeledmonoclonal antibody specifically binding to a tumor antigen for themanufacture of a medicament for the treatment of a patient sufferingfrom a solid tumor overexpressing said tumor antigen previously treatedwith said non-labeled monoclonal antibody and a second monoclonalantibody labeled with a NIR fluorescence label, specifically binding thesame tumor antigen whereby after said previous treatment thefluorescence signal in a region of the solid tumor has decreased by atleast 10%, preferably at least 20%, more preferably at least 30%,compared to the NIR fluorescence signal in said region of the solidtumor before said previous treatment with said non-labeled monoclonalantibody; characterized in that the first and second antibody exhibit nocross reactivity.

Another aspect of the invention is the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa pharmaceutical composition for the treatment of patient suffering froma solid tumor overexpressing said tumor antigen, wherein theadministration pattern of the medicament comprises the following, steps

a) the patient receives a first dose with a second monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to the sametumor antigen;

b) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in a region of the solid tumor ismeasured;

c) the patient receives a first dose with said non-labeled monoclonalantibody;

d) the patient receives a second dose with said second antibody labeledwith a NIR fluorescence label;

e) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in said region of the solid tumoris measured and has decreased by at least 10%, preferably at least 20%,more preferably at least 30%, compared to the signal measured under b);f) the patient receives a second dose with said non-labeled monoclonalantibody based on the result of the measurement of step e);characterized in that the first and second antibody exhibit no crossreactivity.

Another aspect of the invention is the use of a monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to a tumorantigen for the manufacture of a pharmaceutical composition for thetreatment of a patient suffering from a solid tumor overexpressing saidtumor antigen.

Another aspect of the invention is the use of a non-labeled, monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a solid tumor wherein, during saidtreatment, a second monoclonal antibody labeled with a NIR fluorescencelabel, specifically binding to the same tumor antigen is used to monitorthe treatment, characterized in that the first and second antibodyexhibit no cross reactivity.

Another aspect of the invention is a method for acquiring a NIRfluorescence image of a patient suffering from a solid tumoroverexpressing a tumor antigen which has received a dose of a first,non-labeled, monoclonal antibody specifically binding to said tumorantigen and a dose of a second monoclonal antibody labeled with a NIRfluorescence label specifically binding to the same tumor antigen,wherein the NIR fluorescence signal of in a region of the solid tumor ismeasured characterized in that the first and second antibody exhibit nocross reactivity.

Another aspect of the invention is a method for acquiring a NIRfluorescence image wherein the signal of a monoclonal antibody labeledwith a NIR fluorescence label specifically binding to a tumor antigen,in a region of a solid tumor is measured during the treatment of apatient suffering from a solid tumor overexpressing a tumor antigen witha monoclonal antibody specifically binding to the same tumor antigen,characterized in that the first and second antibody exhibit no crossreactivity.

Another aspect of the invention is a method for determining NIRfluorescence signal of a monoclonal antibody labeled with a NIRfluorescence label specifically binding to a tumor antigen in a regionof the solid tumor during the treatment of a patient suffering from asolid tumor overexpressing a tumor antigen with a non-labeled,monoclonal antibody specifically binding to the same tumor antigen,wherein the following steps were performed:

a) the patient receives a first dose with a second monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to the sametumor antigen, wherein the first and second antibody exhibit no crossreactivity,

b) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in a region of the solid tumor ismeasured,

c) the patient receives a first dose with said non-labeled monoclonalantibody

d) the patient receives a second dose with said second antibody labeledwith a NIR fluorescence label,

e) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in said region of the solid tumoris measured and has decreased by at least 10%, preferably at least 20%,more preferably at least 30%, compared to the signal measured under b);f) the patient receives a second dose with said non-labeled monoclonalantibody based on the result of the measurement of step e);characterized in that the first and second antibody exhibit no crossreactivity.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” encompasses the various forms of antibodiesincluding but not being limited to whole antibodies, human antibodies,humanized antibodies and genetically engineered antibodies likemonoclonal antibodies, chimeric antibodies or recombinant antibodies aswell as fragments of such antibodies as long as the characteristicproperties according to the invention are retained.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g. a transgenic mouse, having agenome comprising a human heavy chain transgene and a light human chaintransgene fused to an immortalized cell.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred. Such murine/human chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding murine immunoglobulin variable regions and DNAsegments encoding human immunoglobulin constant regions. Other forms of“chimeric antibodies” encompassed by the present invention are those inwhich the class or subclass has been modified or changed from that ofthe original antibody. Such “chimeric” antibodies are also referred toas “class-switched antibodies.” Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques now well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.5,202,238 and U.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric and bifunctional antibodies.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. Human antibodies are well-known inthe state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr.Opin. Pharmacol. 5 (2001) 368-374). Based on such technology, humanantibodies against a great variety of targets can be produced. Examplesof human antibodies are for example described in Kellermann, S. A., etal., Curr Opin Biotechnol. 13 (2002)593-597.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences in a rearranged form. Therecombinant human antibodies according to the invention have beensubjected to in vivo somatic hypermutation. Thus, the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

As used herein, “specifically binding” refers to an antibodyspecifically binding to the tumor antigen (for which the antibody isspecific). Preferably the binding affinity is of K_(D)-value of 10⁻⁸mol/l or higher (e.g. 10⁻⁹ mol/l), preferably with a K_(D)-value of 10⁻⁹mol/l or higher, more preferably with a K_(D)-value of 10⁻¹⁰ mol/l orhigher. The binding affinity is determined with a standard bindingassay, such as surface plasmon resonance technique (Biacore®).

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The “constant domains” are not involved directly in binding the antibodyto an antigen but are involved in the effector functions (ADCC,complement binding, and CDC).

The “variable region” (variable region of a light chain (VL), variableregion of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the O-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, E. A., et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop”.

A “tumor antigen,” as used herein, includes the meaning known in theart, which includes any molecule expressed on (or associated with thedevelopment of) a tumor cell that is known or thought to contribute to atumorigenic characteristic of the tumor cell. Numerous tumor antigensare known in the art. Whether a molecule is a tumor antigen can also bedetermined according to techniques and assays well known to thoseskilled in the art, such as for example clonogenic assays,transformation assays, in vitro or in vivo tumor formation assays, gelmigration assays, gene knockout analysis, etc. Preferably the term“tumor antigen” when used herein refers to a human transmembrane proteini.e., a cell membrane proteins which is anchored in the lipid bilayer ofcells. The human transmembrane protein will generally comprise an“extracellular domain” as used herein, which may bind a ligand; alipophilic transmembrane domain, a conserved intracellular domaintyrosine kinase domain, and a carboxyl-terminal signaling domainharboring several tyrosine residues which can be phosphorylated. Thetumor antigen include molecules such as EGFR, HER2/neu, HER3, HER4,Ep-CAM, CEA, TRAIL, TRAIL-receptor 1, TRAIL-receptor 2, lymphotoxin-betareceptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R,CTLA-4, fibroblast activation protein (FAP), hepsin, melanoma-associatedchondroitin sulfate proteoglycan (MCSP), prostate-specific membraneantigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1,TIE-2, TNF-alpha, TNF like weak inducer of apoptosis (TWEAK), IL-1R,preferably EGFR, HER2/neu, CEA, CD20, or IGF1-R; more preferablyHER2/neu or EGFR, still more preferably HER2/neu.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors characterized by overexpression of the tumor antigen can bedetermined by standard assays known in the art. Preferablyoverexpression is measured in fixed cells of frozen or paraffin-embeddedtissue sections using immunohistochemical (IHC) detection. When coupledwith histological staining, localization of the targeted protein can bedetermined and extent of its expression within a tumor can be measuredboth qualitatively and semi-quantitatively. Such IHC detection assaysare known in the art and include e.g. the Clinical Trial Assay (CTA),the commercially available LabCorp 4D5 test for the HER2 antigen, andthe commercially available DAKO HercepTest® (DAKO, Carpinteria, Calif.)for the HER2 tumor antigen. The latter assay uses a specific range of 0to 3+ cell staining (0 being normal expression, 3+ indicating thestrongest positive expression) to identify cancers having overexpressionof the HER2 protein (see the Herceptin® (trastuzumab) full prescribinginformation; September 1998; Genentech, Inc., San Francisco, Calif.).Thus, e.g. with regard to the HER2 tumor antigen patients having a solidtumor characterized by overexpression of the HER2 tumor antigen in therange of 1+, 2+, or 3+, preferably 2+ or 3+, more preferably 3+ wouldbenefit from the methods of therapy of the present invention.Alternatively such overexpression can be detected by determination ofthe NIR fluorescence signal in a region of the solid tumor of amonoclonal antibody labeled with a NIR fluorescence label, specificallybinding to said tumor antigen and comparison of said NIR fluorescencesignal or image in a region of the solid tumor to the NIR fluorescencesignal or image of the non-tumorous tissue or other tumorsnon-overexpressing said tumor antigen (see e.g. Examples 1 and 2, FIGS.1 and 2).

The term “the first and second antibody exhibit no cross reactivity”refers to the first non-labeled monoclonal antibody specifically bindingto a tumor antigen and a second monoclonal antibody labeled with a NIRfluorescence label, specifically binding to the same tumor antigenwherein these two antibodies show no cross reactivity with respect tosaid tumor antigen. The cross reactivity of these two antibodies withregards to the same tumor antigen can be detected with the help of acompetitive assay. For this purpose, e.g. with the help of an enzymeimmunoassay, there is tested the extent to which the first antibodycompetes with the second antibody for the binding to an immobilizedtumor antigen. For this purpose, an appropriately immobilized tumorantigen is incubated with the first antibody which conjugated for thepurpose of the assay to a detectable moiety and an excess of the secondantibody. Detectable moieties include direct detectable or indirectdetectable systems. By detection of the bound labeling there can easilybe ascertained the extent to which the antibody in question can displacethe known antibody from the binding site. If there is a displacement ofmore than 10%, preferably of more than 20%, at the same concentration orat higher concentrations, preferably in the case of 105-fold excess ofthe second antibody, referred to the known antibody, then the twoantibodies exhibit cross reactivity. That means that the two antibodiesbind to the same or an overlapping epitope. (See non cross reactive andcross reactive examples e.g. Examples 3, 4 and 5, FIGS. 3, 4 and 5).

By the use of such a first and second antibody which exhibit no crossreactivity a clearly improved (=higher) signal/background ratio isachieved (see e.g. Example 3: 2.88=1440MFI/500MFI—Cy5 labeled pertuzumabafter trastuzumab treatment—FIG. 3b ) compared to the signal/backgroundratio of a first and second antibody which exhibit cross reactivity (seee.g. Example 3: 1.06=530MFI/500MFI—Cy5 labeled trastuzumab aftertrastuzumab treatment FIG. 3a ). This allows a better localization ofthe region of tumor than with the use of cross reactive antibodies; evenif less NIRF labeled antibody is given. Thus by the use of such a firstand second antibody which exhibit no cross reactivity an effective t

Thus in one embodiment of the invention the signal/background ratio bythe use of such a first and second antibody which exhibit no crossreactivity is at least 1.5, preferably at least 2.0.

The abbreviation MFI refers to the mean NIR fluorescence (NIRF) signalintensity [arbitrary units]) NIR fluorescence signal intensity can bequantified by summing up the number and signal intensities of the pixelsin the region of interest (ROI).

The term “Signal/background ratio” refers to the signal/background ratioof the respective NIR fluorescence signal (determined as MFI) in theregion of interest (ROI), e.g., the region of the solid tumor and therespective NIR fluorescence background signal (determined as MFI) e.g.the signal measured in a non-tumor tissue.

In the above competitive assay the directly detectable moietiesconjugated to the first antibody (for assay purposes only) include e.g.chromogens (fluorescent or luminescent groups and dyes), enzymes,NMR-active groups or metal particles, haptens, e.g. digoxigenin, areexamples of detectable labels. Indirect detection systems comprise, forexample, that the detection reagent, e.g., the detection antibody islabeled with a first partner of a bioaffine binding pair. Examples ofsuitable binding pairs are hapten or antigen/antibody, biotin or biotinanalogues such as aminobiotin, iminobiotin or desthiobiotin/avidin orstreptavidin, sugar/lectin, nucleic acid or nucleic acidanalogue/complementary nucleic acid, and receptor/ligand, e.g., steroidhormone receptor/steroid hormone. Preferred first binding pair memberscomprise hapten, antigen and hormone. Especially preferred are haptenslike digoxin and biotin and analogues thereof. The second partner ofsuch binding pair, e.g. an antibody, streptavidin, etc., usually islabeled to allow for direct detection, e.g., by the labels as mentionedabove. The detectable label can also be a photoactivatable cross linkinggroup, e.g. an azido or an azirine group. Metal chelates which can bedetected by electrochemoluminescence are also preferred signal-emittinggroups, with particular preference being given to ruthenium chelates,e.g. a ruthenium (bispyridyl) 32+ chelate. Suitable ruthenium labelinggroups are described, for example, in EP 0 580 979, WO 90/05301, WO90/11511, and WO 92/14138.

Examples of such first and second antibody binding to the same tumorantigen which exhibit no cross reactivity are e.g. the two anti-HER2antibodies trastuzumab and pertuzumab, or the two anti-EGFR antibodiescetuximab and rhMab ICR62. However one skilled in the art can easilygenerate further non-cross reactive antibodies to tumor antigen such asEGFR, HER2/neu, HER3, HER4, Ep-CAM, CEA, TRAIL, TRAIL-receptor 1,TRAIL-receptor 2, lymphotoxin-beta receptor, CCR4, CD19, CD20, CD22,CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activation protein(FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan(MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGFreceptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-alpha, TNF like weakinducer of apoptosis (TWEAK), IL-1R, preferably EGFR, HER2/neu, CEA,CD20, or IGF1-R. For this purpose e.g. phage display techniques can beused (as described in Henderikx et al. Cancer Res. 58 (1998) 4324-4332;Huse et al. Science 246 (1989) 1275-1281 or Kang et al. PNAS 88 (1991)11120-11123) with subsequent chimerization and/or humanization. Alsoimmunization techniques are well known in the art, thus immunizationwith the relevant tumor antigen (e.g. their DNA, the protein orfragments thereof) can be used to generate such antibodies. E.g. humanantibodies to IGF-1R can be prepared according to the followingprocedure:

Generation of a Hybridoma Cell Line Producing Anti-IGF-IR Antibodies

Culture of Hybridomas

Generated HuMab hybridomas are cultured in Hybridoma Express Medium (PAALaboratories GmbH, Austria) supplemented with 2 mM L-glutamine(BioWhittaker) and 4% Origen Cloning Factor (Igen, France) at 37° C. and5% CO₂; or in Iscoves Modified Dulbeco's Medium (500 ml: BioWhittakerEurope, Belgium) supplemented with Fetal Clone Serum (50 ml: Hyclone,Utah), and Origen Hybridoma Cloning Factor (30 ml: Igen, GaithersburgMd.) at 37° C. and 5% CO₂.

Immunization Procedure of Transgenic Mice

Ten HCo7 transgenic mice (4 males and 6 females), strain GG2201(Medarex, San José, Calif., USA) are alternatingly immunized with 1×10⁶NIH 3T3 cells, transfected with an expression vector for IGF-IR, and 20μg soluble extracellular domain of IGF-IR. Six immunizations wereperformed in total, three intraperitoneal (IP) immunizations with theIGF-IR expressing cells and three subcutaneous (SC) immunizations at thetail base with the recombinant protein. For the first immunization, 100μl of 1×10⁶ NIH 3T3 IGF-IR cells is mixed with 100 μl complete Freunds'adjuvant (CFA; Difco Laboratories, Detroit, USA). For all otherimmunizations, 100 μl of cells in PBS were used or recombinant proteinis mixed with 100 μl incomplete Freunds' adjuvant (ICFA; Difco).

Antigen Specific ELISA

Anti-IGF-IR titers in sera of immunized mice are determined by antigenspecific ELISA. IGF-IR soluble extracellular domain at a concentrationof 1 μg/ml in PBS was coated overnight at 4° C., or for two hours at 37°C., to 96 wells plates. Thereafter, the wells were blocked with PBSTC(PBS supplemented with 0.05% Tween®-20 and 2% chicken serum (Gibco BRL))for 1 hour (h) at room temperature. First tap sera were diluted 1/50 inPBSTC, sera from all other taps are pre-diluted 1/100 in PBSTC andserially diluted up to 1/6400. Diluted sera are added to the wells andincubated for 1 h at 37° C. Pre-tap serum is used as negative control.200 ng/ml goat anti-human IGF-IR (100 μg/ml) was used as positivecontrol. Subsequently, plates are washed twice with PBST and incubatedwith horse radish peroxidase (HRP)-conjugated rat anti-human IgG F(ab′)₂(DAKO), diluted 1/2000 in PBSTC for 1 h at 37° C. Wells are washed twicewith PBST and assays were developed with freshly prepared ABTS® solution(1 mg/ml) (ABTS: 2,2′-azino bis(3-ethylbenzthiazoline-6-sulfonic acid)for 30 minutes at room temperature (RT) in the dark. Absorbance ismeasured at 405 nm.

FACS Analysis

In addition to determination by antigen specific ELISA, anti-IGF-IRtiters in sera of immunized mice are also determined by FACS analyses.NIH 3T3 IGF-IR cells and the parental NIH 3T3 cells are incubated withdiluted sera for 30 minutes at 4° C. Alternating IP and SC immunizationswere performed at two weeks intervals starting with an IP immunization.Pre-tap serum (parental NIH 3T3 cells) was used as negative control.Initially, 200 ng/ml goat anti-human IGF-IR was used as positivecontrol. Cells are washed three times in PBS supplemented with 1% bovineserum albumin and 0.01% azide. Subsequently, cells are incubated withfluorescein isothiocyanate (FITC)-conjugated antigen binding fragments(F(ab′)₂ fragments) of rat anti-human human IgG diluted 1/100 in FACSbuffer, for 30 minutes at 4° C. Cells are washed twice in FACS bufferand samples were analyzed on a FACSCalibur (Becton Dickinson,Erembodegem-Aalst, Belgium).

Boosting of Mice

When serum titers of anti-IGF-IR are found to be sufficient, mice areadditionally boosted twice with 15 μg IGF-IR extracellular domain in 200μl PBS intravenously (i.v.) 4 and 3 days before fusion.

Hybridoma Generation

Mice are sacrificed and the spleen and lymph nodes flanking theabdominal aorta and vena cava were collected. Fusion of splenocytes andlymph node cells with the fusion partner SP 2.0 cells are performedaccording to standard operating procedures.

κ-ELISA

To determine whether hybridomas that resulted from the fusion generatehuman antibodies, a κ-ELISA is performed. ELISA plates are coated withrat anti-human IgG κ-light chain antibody (DAKO) diluted 1/10000 in PBSby overnight incubation at 4° C. After discarding the wells, plates areblocked by incubation with PBSTC for 1 hour at room temperature.Thereafter, wells are incubated with hybridoma culture supernatant, ½diluted in PBSTC. Culture medium ½ diluted in PBSTC is used as negativecontrol, κ-light positive mouse serum 1/100 diluted in PBSTC served aspositive control. Subsequently, wells are washed thrice and wereincubated with HRP-conjugated rat anti-human IgG F(ab′)₂ (DAKO), diluted1/2000 in PBSTC for 1 h at 37° C. Wells are washed thrice and assays aredeveloped with freshly prepared ABTS® solution (1 mg/ml) for 30 minutesat room temperature (RT) in the dark. Absorbance are measured at 405 nmin an ELISA plate reader.

In this way anti-IGF-1R antibody libraries can be generated.

All antibodies in such libraries (either generated by this technique orby another immunization or phage display technique) can afterwords betested e.g. with the help of an enzyme immunoassay (see the generaldefinition above and also the example of an IGF-1R Antigen specificELISA above), whether they exhibit cross reactivity towards the sametumor antigen or not. In such a way one skilled in the art can easilygenerate pairs of a first and a second antibody specifically binding tothe same tumor antigen which exhibit no cross reactivity.

For example using this immunization technique in this way it lays in theordinary skills of an artisan to generate a second non-crossreactiveantibody specifically binding to the same tumor antigen as a firstnon-labeled antibody selected from the group consisting of: alemtuzumab,apolizumab, cetuximab, epratuzumab, galiximab, gemtuzumab, ipilimumab,labetuzumab, panitumumab, rituximab, trastuzumab, nimotuzumab,mapatumumab, matuzumab, rhMab ICR62 and pertuzumab. Such secondnon-crossreactive antibody can then be labeled with a NIRF label(e.g.Cy5) and used in one of the embodiments of the invention.

The term “tumor” as used herein refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of tumors include, but are not limited to, carcinoma,lymphoma, blastoma (including medulloblastoma and retinoblastoma),sarcoma (including liposarcoma and synovial cell sarcoma),neuroendocrine tumors (including carcinoid tumors, gastrinoma, and isletcell cancer), mesothelioma, schwannoma (including acoustic neuroma),meningioma, adenocarcinoma, and melanoma.

The term “solid tumors” when used herein refers to tumors selected fromthe group of gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophageal cancer, tumors of the biliary tract, as well as head and neckcancer, preferably breast cancer.

The term “region of a solid tumor” when used herein refers to a zonecomprising the solid tumor. The region of a solid tumor can compriseeither the whole solid tumor or only regional parts of it. The NIRfluorescence signal in the region of said solid tumor is measured, andthe corresponding the NIR fluorescence images are acquired in eithertwo-dimensional or three-dimensional form, e.g. in comparison with thesurrounding non-tumorous tissue or in comparison with NIR fluorescencesignals or images at different time points as a reference.

The terms “co-administration”, “co-administered” or “co-administering”,when used herein, mean that the labeled monoclonal antibody isadministered with the non-labeled monoclonal antibody. Theadministration of the labeled antibody with the non-labeled antibody canbe carried out either as one single formulation or as two separateformulations (one for the non-labeled antibody and one for the labeledantibody). The co-administration can be simultaneous or sequential ineither order, wherein preferably there is a time period while bothmonoclonal antibodies are simultaneously binding to the same tumorantigen on the solid tumor, as long as the tumor is still existent andas long as the tumor overexpresses said tumor antigen. If one singleformulation is used, the two antibodies are co-administeredsimultaneously. If two separate formulations (one for the non-labeledantibody and one for the labeled antibody) are used, the two antibodiesare co-administered either simultaneously (e.g. through one singlecontinuous infusion or through two separate continuous infusions at thesame time) or sequentially. When both antibodies are co-administeredsequentially the labeled antibody can be administered before or afterthe non-labeled antibody either on the same day in two separateadministrations, or e.g. the labeled antibody is administered on day 1for acquiring a NIR fluorescence image and the non-labeled antibody isco-administered afterward e.g. on day 2 to day 7. Then after a certainperiod e.g. from one to 5 weeks, which may include furtheradministration of the non-labeled antibody, the antibody labeled with aNIR fluorescence label is administered again for acquiring a NIRfluorescence image. Based on the comparison of these NIR fluorescenceimages which are preferably acquired in the region of the solid tumorusing the same conditions (e.g. same amount of labeled antibody, sametime point after administration, same acquisition time, etc.), a furtheradministration of the non-labeled antibody will be given when the NIRfluorescence signal has decreased at least 10%, preferably at least 20%and more preferably at least 30%. The term “dose” when used hereinrefers to the administration of the monoclonal antibodies. The “dose” ofthe non-labeled monoclonal antibodies, can be e.g. in the range fromabout 0.05 mg/kg to about 10 mg/kg body weight. Thus, one or moreconsecutive doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kgbody weight may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every two or threeweeks, (e.g. such that the patient receives from about two to abouttwenty, e.g. about six doses of trastuzumab and pertuzumab each). Also,an initial higher loading dose, followed by one or more lower doses maybe administered. The monoclonal antibody labeled with a NIR fluorescencelabel in is hereby co-administered in an amount or dose of at least0.001 mg/kg body weight, preferably at least 0.01 mg/kg body weight,more preferably at least 0.01 mg/kg body weight. The exact amount or“dose” can vary and depends e.g. on the label and his quantum yield. Theamount or “dose” of the non-labeled monoclonal antibody and themonoclonal antibody labeled with a NIR fluorescence label can be definedby the skilled artisan by simple routine experiments.

The term “administration pattern of the medicament” when used hereinrefers to the preferably sequential steps during administration of saidmedicament, which may include the administrations itself, measurement ofNIR fluorescence signals in the region of the solid tumor, comparison ofdifferent NIR fluorescence signals in the region of the same solid tumormeasured under the same conditions e.g. before and after administrationof the non-labeled monoclonal antibody, administration of a furtherdoses of the non-labeled monoclonal antibody base on the finding thatthe NIR fluorescence signals in the region of the same solid tumor hasdecreased during the treatment.

The term “during said treatment” with a first non-labeled monoclonalantibody when used herein refers to the co-administration of secondmonoclonal antibody labeled with a NIR fluorescence label which can besimultaneous or sequential in either order, wherein preferably there isa time period while both monoclonal antibodies are simultaneouslybinding to the same tumor antigen on the solid tumor, as long as thetumor is still existent and as long as the tumor expresses oroverexpresses said tumor antigen. In connection with the term “duringsaid treatment” with a first non-labeled monoclonal antibody thetreatment period of said first non-labeled monoclonal antibody starts atthe first dose administration, (and can include several consecutiveadministrations of said first antibody, while there is always an amount(preferably a therapeutically effective amount) of said antibody presentin the patient) and ends at the time when after the last administrationof said first antibody said first antibody was fully degraded withinsaid patient (preferably when said first antibody was degraded until anresidual amount which below the therapeutically effective amount).

The term “patient suffering from a solid tumor overexpressing said tumorantigen previously treated with said non-labeled monoclonal antibody”when used herein means that said at least one dose of said non-labeledmonoclonal antibody has been administered to said patient before thetreatment of the patient with both monoclonal antibodies.

It is self-evident that the non-labeled antibody administered to thepatient in a therapeutically effective amount which is the amount of thesubject compound or combination that will elicit the biological ormedical response of a tissue, system, animal or human that is beingsought by the researcher, veterinarian, medical doctor or otherclinician.

As used herein, the term “patient” preferably refers to a human in needof treatment to treat cancer, or a precancerous condition or lesion.However, the term “patient” can also refer to non-human animals,preferably mammals such as dogs, cats, horses, cows, pigs, sheep andnon-human primates, among others, that are in need of treatment.

The terms “antibody labeled with a NIR fluorescence label”, “labeledantibody” or “labeled monoclonal antibody” as used herein refer tomonoclonal antibodies which are conjugated to NIR fluorescence label.Conjugation techniques have significantly matured during the past yearsand an excellent overview is given in Aslam, M., and Dent, A.,Bioconjugation, London (1998) 216-363, and in the chapter “Macromoleculeconjugation” in Tijssen, P. “Practice and theory of enzyme immunoassays”(1990) 221-278 Elsevier, Amsterdam.

The term “non-labeled antibody” as used herein refers to a monoclonalantibody which is not labeled to a NIR fluorescence label nor conjugatedto another moiety.

The term “NIR” as used herein means near-infrared.

The invention comprises a pharmaceutical composition comprising

-   -   a) a non-labeled monoclonal antibody specifically binding to a        tumor antigen and    -   b) a second monoclonal antibody labeled with a NIR fluorescence        label, specifically binding to the same tumor antigen,    -   characterized in that, the first and second antibody exhibit no        cross reactivity.

Such composition can be composed of either one compartment comprisingboth antibodies in on single formulation or of two compartments, onecomprising the non-labeled monoclonal antibody in a first formulationand one comprising the labeled monoclonal antibody in a secondformulation. Such composition is intended for the co-administration ofsuch non-labeled monoclonal antibody and labeled monoclonal antibody.

In one embodiment, said tumor antigen is selected from the groupconsisting of EGFR, HER2/neu, HER3, HER4, Ep-CAM, CEA, TRAIL,TRAIL-receptor 1, TRAIL-receptor 2, lymphotoxin-beta receptor, CCR4,CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblastactivation protein (FAP), hepsin, melanoma-associated chondroitinsulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA),VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2,TNF-alpha, TNF like weak inducer of apoptosis (TWEAK), IL-1R, preferablyEGFR, HER2/neu, CEA, CD20, or IGF1-R; more preferably HER2/neu or EGFR,still more preferably HER2/neu.

In another embodiment, the non-labeled monoclonal antibody is ananti-HER2 antibody, preferably trastuzumab or pertuzumab, morepreferably trastuzumab.

In another embodiment, the non-labeled monoclonal antibody is ananti-EGFR antibody, preferably cetuximab, rhMab ICR62, nimotuzumab, ormatuzumab, more preferably cetuximab or rhMab ICR62.

In another embodiment, the non-labeled monoclonal antibody is ananti-IGF 1R antibody.

In another embodiment, the non-labeled monoclonal antibody is selectedfrom the group of:

alemtuzumab, apolizumab, cetuximab, epratuzumab, galiximab, gemtuzumab,ipilimumab, labetuzumab, panitumumab, rituximab, trastuzumab,nimotuzumab, mapatumumab, matuzumab, rhMab ICR62 and pertuzumab,preferably trastuzumab, cetuximab, rhMab ICR62 and pertuzumab, morepreferably trastuzumab.

The composition typically comprises the antibody labeled with a NIRfluorescence label in an amount of at least 0.001 mg/kg body weight,preferably at least 0.01 mg/kg body weight, more preferably at least0.01 mg/kg body weight. The exact amount can vary and depends e.g. onthe label and his quantum yield. The amount can be defined by theskilled artisan by simple routine experiments.

Trastuzumab (sold under the trade name Herceptin®) is a recombinanthumanized anti-HER2 monoclonal antibody used for the treatment of HER2over-expressed/HER2 gene amplified metastatic breast cancer. Trastuzumabbinds specifically to the same epitope of HER2 as the murine anti-HER2antibody 4D5 described in Hudziak, R. M., et al., Mol. Cell. Biol. 9(1989) 1165-1172. Trastuzumab is a recombinant humanized version of themurine anti-HER2 antibody 4D5, referred to as rhuMAb 4D5 or trastuzumab)and has been clinically active in patients with HER2-overexpressingmetastatic breast cancers that had received extensive prior anticancertherapy. (Baselga, J., et al, J. Clin. Oncol. 14 (1996) 737-744).Trastuzumab and its method of preparation are described in U.S. Pat. No.5,821,337.

Pertuzumab (Omnitarg®) is another recombinant humanized anti-HER2monoclonal antibody used for the treatment of HER2 positive cancers.Pertuzumab binds specifically to the 2C4 epitope, a different epitope onthe extracellular domain of HER2 as trastuzumab. Pertuzumab is the firstin a new class of HER dimerisation inhibitors (HDIs). Through itsbinding to the HER2 extracellular domain, pertuzumab blocksligand-activated heterodimerisation of HER2 with other HER familymembers, thereby inhibiting downstream signalling pathways and cellularprocesses associated with tumor growth and progression (Franklin, M. C.,et al. Cancer Cell 5 (2004) 317-328 and Friess, T, et al. Clin CancerRes 11 (2005) 5300-5309). Pertuzumab is a recombinant humanized versionof the murine anti-HER2 antibody 2C4 (referred to as rhuMAb 2C4 orpertuzumab) and it is described together with the respective method ofpreparation in WO 01/00245 and WO 2006/007398.

Trastuzumab and pertuzumab are examples of first and second monoclonalantibodies specifically binding to the same tumor antigen, characterizedin that, the first and second antibody exhibit no cross reactivity.Further examples include the two anti-EGFR antibodies, cetuximab andrhMab ICR62.

Cetuximab is chimeric monoclonal anti-EGFR antibody 225 (c MAb 225, U.S.Pat. No. 4,943,533 and EP 0359 282), for use in the treatment of EGFRexpressing tumors. The C225 antibody (Cetuximab) was demonstrated toinhibit EGF-mediated tumor cell cascade.

The rhMab ICR62, another anti-EGFR antibody, is an recombinant humanizedversion of the rat ICR62 antibody and is described in WO 2006/082515.

The two anti-EGFR antibodies, cetuximab and rhMab ICR62 representfurther examples of first and second monoclonal antibodies specificallybinding to the same tumor antigen, characterized in that, the first andsecond antibody exhibit no cross reactivity.

Another aspect of the invention is the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a patient suffering from a solid tumoroverexpressing said tumor antigen

wherein the non-labeled monoclonal antibody is co-administered with asecond monoclonal antibody labeled with a NIR fluorescence label,specifically binding to the same tumor antigen, characterized in thatthe first and second antibody exhibit no cross reactivity.

In one aspect of the invention the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a patient suffering from a solid tumoroverexpressing said tumor antigen is characterized in that a NIRfluorescence image of said patient is acquired.

In another aspect of the invention such use is characterized in that theNIR fluorescence signal of said second monoclonal antibody labeled witha NIR fluorescence label, specifically binding to the same tumor antigenin a region of the solid tumor is measured.

Another aspect of the invention is the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a patient suffering from a solid tumoroverexpressing said tumor antigen previously treated with saidnon-labeled monoclonal antibody and a second monoclonal antibody labeledwith a NIR fluorescence label, specifically binding the same tumorantigen whereby after said previous treatment the NIR fluorescencesignal in a region of the solid tumor has decreased by at least 10%,preferably at least 20%, more preferably at least 30%, compared to theNIR fluorescence signal in said region of the solid tumor before saidprevious treatment with said non-labeled monoclonal antibody;

characterized in that the first and second antibody exhibit no crossreactivity.

Another aspect of the invention is the use of a non-labeled monoclonalantibody specifically binding a tumor antigen for the manufacture of amedicament for the treatment of a patient suffering from a solid tumoroverexpressing said tumor antigen wherein the administration pattern ofthe medicament comprises the following steps:

a) the patient receives a first dose with said non-labeled monoclonalantibody;

b) the NIR fluorescence signal of a second antibody labeled with a NIRfluorescence label, specifically binding to the same tumor antigen,after said first dose with said non-labeled monoclonal antibody hasdecreased by at least 10%, preferably at least 20%, more preferably atleast 30%, in a region of the solid tumor, compared to the NIRfluorescence signal in said region of the solid tumor, before said firstdose with said non-labeled monoclonal antibody;c) the patient receives a second dose with said non-labeled monoclonalantibody based on the result of the measurement of step b);characterized in that the first and second antibody exhibit no crossreactivity.

Preferably the steps a) to c) are carried out as consecutive steps a),b), and c).

Another aspect of the invention is the use of a non-labeled monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of patient suffering from a solid tumoroverexpressing said tumor antigen, wherein the administration pattern ofthe medicament comprises the following steps:

a) the patient receives a first dose with a second monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to the sametumor antigen;

b) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in a region of the solid tumor ismeasured;

c) the patient receives a first dose with said non-labeled monoclonalantibody;

d) the patient receives a second dose with said second antibody labeledwith a NIR fluorescence label;

e) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in said region of the solid tumoris measured and has decreased by at least 10%, preferably at least 20%,more preferably at least 30%, compared to the signal measured under b);f) the patient receives a second dose with said non-labeled monoclonalantibody based on the result of the measurement of step e);characterized in that the first and second antibody exhibit no crossreactivity.

Preferably the steps a) to f) are carried out as consecutive steps a),b), c), d), e) and f).

Another aspect of the invention is the use of a monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to a tumorantigen for the manufacture of a pharmaceutical composition for thetreatment of a patient suffering from a solid tumor overexpressing saidtumor antigen.

Another aspect of the invention is the use of a non-labeled, monoclonalantibody specifically binding to a tumor antigen for the manufacture ofa medicament for the treatment of a solid tumor overexpressing saidtumor antigen wherein a second monoclonal antibody labeled with a NIRfluorescence label, specifically binding to the same tumor antigen isused to determine the response to said treatment, characterized in thatthe first and second antibody exhibit no cross reactivity.

The term “to determine the response to said treatment” when used hereinrefers to the acquisition of the NIR fluorescence signals or imagesduring the treatment of the patient suffering from a solid tumoroverexpressing a tumor antigen with a non-labeled monoclonal antibody.E.g. several measurements in a region the solid tumor at different timepoints of the treatment can be performed. Or several NIR fluorescenceimages of the patient suffering from a solid tumor overexpressing atumor antigen can be acquired. The “response” to the treatment can thenbe categorized for solid tumors overexpressing said tumor antigen independency of the decrease (increase) of the NIR fluorescence signal insaid region of the solid tumor, which correlates to an decrease(increase) of the expression of tumor antigen in said region of thesolid tumor (see e.g. Example 4, FIG. 4). In this connection anotherpreferred aspect of the invention is the use of a non-labeled,monoclonal antibody specifically binding to a tumor antigen for themanufacture of a medicament for the treatment of a solid tumor wherein asecond monoclonal antibody labeled with a NIR fluorescence label,specifically binding to the same tumor antigen is used to determine theexpression of said tumor antigen, characterized in that the first andsecond antibody exhibit no cross reactivity.

Preferably the uses of a non-labeled monoclonal antibody specificallybinding to a tumor antigen for the manufacture of a pharmaceuticalcomposition as described above as well as the methods described beloware characterized in that said tumor antigen, against which bothmonoclonal antibodies are specifically binding, is selected from thegroup consisting of EGFR, HER2/neu, HER3, HER4, Ep-CAM, CEA, TRAIL,TRAIL-receptor 1, TRAIL-receptor 2, lymphotoxin-beta receptor, CCR4,CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblastactivation protein (FAP), hepsin, melanoma-associated chondroitinsulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA),VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2,TNF-alpha, TNF like weak inducer of apoptosis (TWEAK), IL-1R, preferablyEGFR, HER2/neu, CEA, CD20, or IGF-1-R; more preferably HER2/neu.

Another aspect of the invention is a method for acquiring a NIRfluorescence image of a patient suffering from a solid tumoroverexpressing a tumor antigen which has received a dose of a first,non-labeled, monoclonal antibody specifically binding to said tumorantigen and a dose of a second monoclonal antibody labeled with a NIRfluorescence label, specifically binding to the same tumor antigen,wherein the NIR fluorescence signal of in a region of the solid tumor ismeasured characterized in that the first and second antibody exhibit nocross reactivity.

Another aspect of the invention is a method for acquiring a NIRfluorescence image wherein the signal of a monoclonal antibody labeledwith a NIR fluorescence label, specifically binding to a tumor antigen,in a region of a solid tumor is measured during the treatment of apatient suffering from a solid tumor overexpressing a tumor antigen witha monoclonal antibody specifically binding to the same tumor antigen,characterized in that the first and second antibody exhibit no crossreactivity.

Another aspect of the invention is a method for determining NIRfluorescence signal of a monoclonal antibody labeled with a NIRfluorescence label, specifically binding to a tumor antigen in a regionof the solid tumor during the treatment of a patient suffering from asolid tumor overexpressing a tumor antigen with a non-labeled,monoclonal antibody specifically binding to the same tumor antigen,wherein the following steps are performed:

a) the patient receives a first dose with a second monoclonal antibodylabeled with a NIR fluorescence label, specifically binding to the sametumor antigen, wherein the first and second antibody exhibit no crossreactivity,

b) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in a region of the solid tumor ismeasured,

c) the patient receives a first dose with said non-labeled monoclonalantibody

d) the patient receives a second dose with said second antibody labeledwith a NIR fluorescence label,

e) the NIR fluorescence signal of said second monoclonal antibodylabeled with a NIR fluorescence label in said region of the solid tumoris measured and has decreased by at least 10%, preferably at least 20%,more preferably at least 30%, compared to the signal measured under b);f) the patient receives a second dose with said non-labeled monoclonalantibody based on the result of the measurement of step e);characterized in that the first and second antibody exhibit no crossreactivity.

Preferably the steps a) to f) are carried out as consecutive steps a),b), c), d), e) and f).

Preferably in such methods the signal/background ratio is at least 1.5,preferably 2.0.

Said “monoclonal antibody labeled with a NIR fluorescence label” islabeled with near infrared (NIR) fluorescence label suitable foracquiring of a NIR fluorescence image in the region of the solid tumor.

NIR fluorescence labels with excitation and emission wavelengths in thenear infrared spectrum are used, i.e., 640-1300 nm preferably 640-1200nm, and more preferably 640-900 nm. Use of this portion of theelectromagnetic spectrum maximizes tissue penetration and minimizesabsorption by physiologically abundant absorbers such as hemoglobin(<650 nm) and water (>1200 nm). Ideal near infrared fluorochromes for invivo use exhibit:

(1) narrow spectral characteristics,

(2) high sensitivity (quantum yield),

(3) biocompatibility, and

(4) decoupled absorption and excitation spectra.

Various near infrared (NIR) fluorescence labels are commerciallyavailable and can be used to prepare probes according to this invention.Exemplary NIRF labels include the following: Cy5.5, Cy5 and Cy7(Amersham, Arlington Hts., IL; IRD41 and IRD700 (LI-COR, Lincoln,Nebr.); NIR-1, (Dejindo, Kumamoto, Japan); LaJolla Blue (Diatron, Miami,Fla.); indocyanine green (ICG) and its analogs (Licha, K., et al., Proc.SPIE-Int. Soc. Opt. Eng. 2927 (1996) 192-198; Ito et al., U.S. Pat. No.5,968,479); indotricarbocyanine (ITC; WO 98/47538); and chelatedlanthanide compounds. Fluorescent lanthanide metals include europium andterbium. Fluorescence properties of lanthanides are described inLackowicz, Principles of Fluorescence Spectroscopy 2nd Ed. KluwarAcademic New York (1999).

Accordingly, said “monoclonal antibody labeled with a NIR fluorescencelabel” is preferably labeled by a NIR fluorescence label selected fromthe group of Cy5.5, Cy5, Cy7, IRD41, IRD700, NIR-1, LaJolla Blue,indocyanine green (ICG), indotricarbocyanine (ITC) and SF64, 5-29, 5-36and 5-41 (from WO 2006/072580), more preferably said antibody is labeledwith a NIRF label selected from the group of Cy5.5, Cy5 and Cy7.

The methods used for coupling of the NIR fluorescence labels are wellknown in the art. The conjugation techniques of NIR fluorescence labelsto an antibody have significantly matured during the past years and anexcellent overview is given in Aslam, M., and Dent, A., Bioconjugation(1998) 216-363, London, and in the chapter “Macromolecule conjugation”in Tijssen, P., “Practice and theory of enzyme immunoassays” (1990),Elsevier, Amsterdam.

Appropriate coupling chemistries are known from the above citedliterature (Aslam, supra). The NIR fluorescence label, depending onwhich coupling moiety is present, can be reacted directly with theantibody either in an aqueous or an organic medium. The coupling moietyis a reactive group or activated group which is used for chemicallycoupling of the fluorochrome label to the antibody. The fluorochromelabel can be either directly attached to the antibody or connected tothe antibody via a spacer to form a NIR fluorescence label conjugatecomprising the antibody and a NIR fluorescence label. The spacer usedmay be chosen or designed so as to have a suitably long in vivopersistence (half-life) inherently.

“Measurement” or “determining” of the NIR fluorescence signal in aregion the solid tumor is performed after administration of the labeledantibody to the patient. Or, if the composition according to theinvention is used, after the administration of the composition of thenon-labeled antibody and the labeled antibody to the patient. Themeasurement can be performed on defined time points afteradministration, e.g., 1 day, 2 days or 3 or even more days or any othertime point appropriate for acquiring a comparable NIR fluorescencesignal or image in a region the solid tumor. The duration of themeasurement or the time point after administration can be adjusted by aperson skilled in the art in a way to get an appropriate NIRfluorescence signal or image.

For the NIR fluorescence measurement different devices and techniquescan be used, e.g. for external solid tumors like breast tumors, aSoftScan® apparatus from ART Advanced Research Technologies Inc.(http://www.art.ca/en/products/softscan.html) is suitable (Intes X,Acad. Radiol. 12 (2005) 934-947) For internal disease areas, likecolorectal or lung cancer endoscopic techniques or a combination ofmicrosurgery-endoscopy can be used.

An imaging system for NIR fluorescence measurement useful in thepractice of this invention typically includes three basic components:(1) a near infrared light source, (2) a means for separating ordistinguishing fluorescence emissions from light used for fluorochromeexcitation, and (3) a detection system.

The light source provides monochromatic (or substantially monochromatic)near infrared light. The light source can be a suitably filtered whitelight, i.e., bandpass light from a broadband source. For example, lightfrom a 150-watt halogen lamp can be passed through a suitable bandpassfilter commercially available from Omega Optical (Brattleboro, Vt.). Insome embodiments, the light source is a laser. See, e.g., Boas, D. A.,et al., 1994, Proc. Natl. Acad. Sci. USA 91 4887-4891; Ntziachristos,V., et al., Proc. Natl. Acad. Sci. USA 97 2000 2767-2772; Alexander, W.,1991, J. Clin. Laser Med. Surg. 9 416-418.

A high pass filter (700 nm) can be used to separate fluorescenceemissions from excitation light. A suitable high pass filter iscommercially available from Omega optical.

In general, the light detection system can be viewed as including alight gathering/image forming component and a light detection/imagerecording component. Although the light detection system may be a singleintegrated device that incorporates both components, the lightgathering/image forming component and light detection/image recordingcomponent will be discussed separately.

A particularly useful light gathering/image forming component is anendoscope. Endoscopic devices and techniques that have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen, J., et al., J. Photochem. Photobiol. B 52 (1999)131-135), ovarian cancer (Major, A. L., et al., Gynecol. Oncol. 66(1997) 122-132), colon (Mycek, M. A., et al., Gastrointest. Endoscopy.48 (1998) 390-394; Stepp, H., et al., Endoscopy 30 (1998) 379-386) bileducts (Izuishi, K., et al., Hepatogastroenterology 46 (1999) 804-807),stomach (Abe, S., et al., Endoscopy 32 (2000) 281-286), bladder(Kriegmair, M., et al., Urol. Int. 63 (1999) 27-31; Riedl, C. R., etal., J. Endourol. 13 755-759), and brain (Ward, H. A., J. Laser Appl. 10(1998) 224-228) can be employed in the practice of the presentinvention.

Other types of light gathering components useful in the invention arecatheter-based devices, including fiber optics devices. Such devices areparticularly suitable for intravascular imaging. See, e.g., Tearney, G.J., et al., Science 276 (1997) 2037-2039; Boppart, S. A., Proc. Natl.Acad. Sci. USA 94 (1997) 4256-4261.

Still other imaging technologies, including phased array technology(Boas, D. A., et al., Proc. Natl. Acad. Sci. 19 USA 91 (1994)4887-4891;Chance, B., Ann. NY Acad. Sci. 838 (1998) 29-45), diffuse opticaltomography (Cheng, X., et al., Optics Express 3 (1998) 118-123; Siegel,A., et al., Optics Express 4 (1999) 287-298), intravital microscopy(Dellian, M., et al., Br. LT. Cancer 82 (2000) 1513-1518; Monsky, W. L.,et al., Cancer Res. 59 (1999) 4129-4135; Fukumura, D., et al., Cell 94(1998) 715-725), and confocal imaging (Korlach, J., et al., Proc. Natl.Acad. Sci. USA 96 (1999) 8461-8466; Rajadhyaksha, M., et al., J. Invest.Dermatol. 104 (1995)946-952; Gonzalez, S., et al., J. Med. 30 (1999)337-356) can be employed in the practice of the present invention.

Any suitable light detection/image recording component, e.g., chargecoupled device (CCD) systems or photographic film, can be used in theinvention. The choice of light detection/image recording will depend onfactors including type of light gathering/image forming component beingused. Selecting suitable components, assembling them into a nearinfrared imaging system, and operating the system is within ordinaryskill in the art.

Useful apparatuses for acquiring a NIR fluorescence image are e.g. theSoftScan® apparatus from ART Advanced Research Technologies Inc.; ImageStation In-Vivo F; Image Station In-Vivo FX; In-Vivo Imaging System FXPro from Molecular Imaging Systems, Carestream Health, Inc. (formerlyKodak Molecular Imaging Systems); Aerius™ Automated Infrared ImagingSystem and Odyssey Infrared Imaging System™ from LI-COR Biosciences; LB983 NightOWL II from BERTHOLD TECHNOLOGIES or other appropriate devices.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 NIR fluorescence images of different solid tumors overexpressingand not overexpressing a HER2 tumor antigen:

-   -   In mice with    -   a) BT474 s.c (subcutaneous). model, a strongly HER2        overexpressing tumor model (3+ according to DAKO HercepTest®)        (FIG. 1a ),    -   b) a A549 s.c. model, a tumor model with no HER2 overexpression        (0 according to DAKO HercepTest®) (FIG. 1b ) and    -   c) in tumor free mice (FIG. 1c ), pertuzumab labeled with Cy5        was injected i.v. at a single dose of 20 microgram per mouse and        NIR fluorescence signal was measured after 24 h. Acquisition        time was 2 seconds. The NIR fluorescence images indicate: in        FIG. 1a that i) the BT474 tumor cells overexpress HER2 tumor        antigen and ii) a significant NIR fluorescence signal in the        region of the solid tumor can be measured compared to the        non-tumorous tissue; in FIG. 1b that i) the A549 tumor cells        show no significant overexpression of the HER2 tumor antigen        and ii) no significant NIR fluorescence signal in the region of        the solid tumor can be detected compared to the non-tumorous        tissue; in FIG. 1c that i) the non-tumorous cells in the        tumor-free mice show no significant overexpression of the HER2        tumor antigen and ii) no significant NIR fluorescence signal can        be detected.

FIG. 2 NIR fluorescence images of solid tumors overexpressing HER2 tumorantigen:

-   -   In four mice with a KPL4 model, a strongly HER2 overexpressing        tumor model (3+ according to DAKO HercepTest®), trastuzumab        labeled with Cy5 (FIG. 2a ) was injected i.v. in two mice, and        pertuzumab labeled with Cy5 (FIG. 2b ) was injected i.v. in the        other two mice, all at a single dose of 50 microgram per mouse        and NIR fluorescence signal was measured after 24 h. Acquisition        time was 4 seconds. The NIR fluorescence images indicate that i)        the KPL4 tumor cells overexpress HER2 tumor antigen and ii) a        significant NIR fluorescence signal in the region of the solid        tumor can be measured compared to the non-tumorous tissue using        either trastuzumab labeled with Cy5 (FIG. 2a ) or pertuzumab        labeled with Cy5 (FIG. 2b ).

FIG. 3 NIR fluorescence images of solid tumors overexpressing HER2 tumorantigen during the treatment of a patient with non-labeled trastuzumab(early phase-48 h after first trastuzumab application)—use of pertuzumablabeled with Cy5 (no cross reactivity with trastuzumab) for NIRfluorescence imaging

-   -   In a KPL4 model, a strongly HER2 overexpressing tumor model (3+        according to DAKO HercepTest®), in a first administration        non-labeled trastuzumab was injected i.p. at a single dose of 30        mg/kg, and 48 h later in a second administration either        trastuzumab labeled with Cy5 (FIG. 3a ) or pertuzumab labeled        with Cy5 (FIG. 3b ) was injected i.v. at a single dose of 50        microgram per mouse and NIR fluorescence signal was measured        after 24 h after the second administration. Acquisition time was        4 seconds. The NIR fluorescence images indicate that i) during        the treatment of a solid tumor overexpressing HER2 tumor antigen        with trastuzumab (or a patient suffering from a solid tumor        overexpressing HER2 tumor antigen), trastuzumab labeled with Cy5        (which is cross reactive with the non-labeled trastuzumab) is        not suitable for the detection of a significant NIR fluorescence        signal in the region of the solid tumor and (FIG. 3a ); ii)        during the treatment of a solid tumor overexpressing HER2 tumor        antigen with trastuzumab, pertuzumab labeled with Cy5 (which        exhibit no cross reactivity with non-labeled trastuzumab) is        suitable for the detection of a significant NIR fluorescence        signal in the region of the solid tumor (FIG. 3b )

FIG. 4 NIR fluorescence images of solid tumors overexpressing HER2 tumorantigen during the weekly treatment with non-labeled pertuzumab (aftersecond pertuzumab application)—use of trastuzumab labeled with Cy5 (nocross reactivity with pertuzumab) for NIR fluorescence imaging

-   -   In the KPL-4 model, pertuzumab was injected i.p. twice weekly.        The first injection (loading dose) was 30 mg/kg and the second        application (maintenance dose) was 15 mg/kg. Control animals        received PBS. After 48 hours after the second administration        mice of both groups were injected with trastuzumab labeled with        Cy5 and 4 days later NIR fluorescence signal was measured using        an acquisition time of 3 seconds. The NIR fluorescence images        indicate that i) that that the NIR fluorescence signal in the        pertuzumab treated mice (FIG. 4a ) has decreased compared to the        NIR fluorescence signal in the PBS treated mice (FIG. 4b ),        and ii) thus, at the solid HER2overexpressing KPL-4 tumor a        response to the treatment with pertuzumab specifically binding        to HER2 is detectable.

FIG. 5 NIR fluorescence imaging of solid tumors expressing EGFR tumorantigen during the treatment with non-labeled cetuximab—use of rhMabICR62 labeled with Cy5 (no cross reactivity with cetuximab) for NIRfluorescence imaging

-   -   Female Balb/c nude mice carrying Calu3 tumors were injected i.p.        with once weekly for four weeks at a dose of 2.5 mg/kg        Cetuximab. Two days after the last treatment mice received 2        mg/kg Cy5 labeled rhMab ICR62. NIR fluorescence signal was        measured 24 hours thereafter with an acquisition time of 4        seconds. The results demonstrate that pre-injection with        non-labeled cetuximab allows subsequent detection of EGFR        expressing tumor cells and exact localization of the tumor        tissue when Cy5 labeled rhMab ICR62 is applied. This indicates        that rhMab ICR62 binds to an epitope which differs from the        epitope recognized by cetuximab, and shows that cetuximab and        rhICR62 exhibit no cross reactivity against the same EGFR tumor        antigen. Thus the NIR fluorescence image indicates that that at        the solid EGFR expressing Calu3 tumor a response to the        treatment with cetuximab specifically binding to HER2 is        detectable with the non-crossreactive CY5 labeled rhMab ICR62.

EXAMPLES

Introduction

The current study examined the NIR fluorescence imaging of monoclonalantibodies labeled with a NIR fluorescence label, specifically binding atumor antigen alone or after or during a previous treatment with anon-labeled monoclonal antibody specifically binding to same tumorantigen (wherein the first and second antibody exhibit no crossreactivity) in different human xenograft models in which said tumorantigen was overexpressed or not overexpressed.

Cell Lines and Culture Conditions

The human breast cancer cell line KPL-4, kindly provided by J.Kureabashi, has been established from the malignant pleural effusion ofa breast cancer patient with an inflammatory skin metastasis andoverexpresses ErbB family receptors. (Kurebayashi, J., et al., Br. J.Cancer 79 (1999) 707-17) Tumor cells are routinely cultured in DMEMmedium (PAA Laboratories, Austria) supplemented with 10% fetal bovineserum (PAA) and 2 mM L-glutamine (Gibco) at 37° C. in a water-saturatedatmosphere at 5% CO2. Culture passage is performed with trypsin/EDTA 1×(PAA) splitting twice/week. Cell passage P6 was used for the in vivostudy.

The human breast cancer cell line BT474, which overexpresses Her2 wasobtained from ATCC. The cells were grown in vitro in RPMI 1640 Mediumcontaining 10% FBS (PAA), 2 mM L-Glutamine, 1 mM Na-Pyruvat and 10 mMHepes at 37° C. in a water-saturated atmosphere at 5% CO2. The cells ofthe third passage were used for subcutaneous injection into the mice.

The Her2 negative human A549 lung carcinoma cells were obtained from theDSMZ. The cells were routinely cultured using the same protocol as forthe BT474 cell line, but without the addition of Hepes. Culture passageis performed with trypsin/EDTA 1× (PAA) splitting twice/week. Cellpassage P3 was used for the in vivo study.

The EGF1R positive human Calu3 lung tumor cells were obtained fromRoche, Kamakura. The cells were grown in vitro in Eagle's MEM withEarle's BSS, 10% FCS, 1 mM Na-pyruvat and 0.1 mM NEAA at 37° C. in awater-saturated atmosphere at 5% CO2. The cells of the third passagewere used for subcutaneous injection into the mice.

Animals

Female SCID beige (C.B.-17) mice and female Balb/c nude mice; age 10-12weeks; body weight 18-20 g (Charles River, Sulzfeld, Germany) aremaintained under specific-pathogen-free condition with daily cycles of12 h light/12 h darkness according to international guidelines(GV-Solas; Felasa; TierschG). After arrival, animals are housed in thequarantine part of the animal facility for one week to get accustomed tonew environment and for observation. Continuous health monitoring iscarried out on regular basis. Diet food (Alltromin) and water (acidifiedpH 2.5-3) are provided ad libitum.

Tumor Cell Injection

Tumor cells were harvested (trypsin-EDTA) from culture flasks (GreinerTriFlask) and transferred into 50 ml culture medium, washed once andresuspended in PBS. After an additional washing step with PBS andfiltration (cell strainer; Falcon 100 μm) the final cell titer wasadjusted appropriately. Tumor cell suspension was carefully mixed withtransfer pipette to avoid cell aggregation. Anesthesia was performedusing a Stephens's inhalation unit for small animals with preincubationchamber (plexiglas), individual mouse nose-mask (silicon) and Isoflurane(Pharmacia-Upjohn, Germany) in a closed circulation system. Two daysbefore injection the fur of the animals was shaved.

KPL-4 tumor cells (3×10e6 in 20 μl PBS) were injected orthotopicallyinto the right penultimate inguinal mammary fat pad (i.m.f.p.) of eachanesthetized mouse. For this orthotopic implantation, the cellsuspension was injected through the skin under the nipple. Tumor cellinjection corresponds to day 1 of the experiment.

BT474 tumor cells (5×10e6) were injected in 100 μl Matrigel (BectonDickinson) subcutaneously into the right flank of the animals. 17 betaestradiol was supplemented via the drinking water. Initially, from day−1 until day 33 of the study the drinking water was supplemented with2.5 μg 17 beta estradiol/ml water.

A549 tumor cells (1×10e7 in 100 μl PBS) were injected subcutaneouslyinto the right flank of the animals.

Calu3 tumor cells (5×10e6 in 100 μl PBS) were injected subcutaneouslyinto the right flank of the animals.

Monitoring of Clinical Symptoms and Body Weight

Animals were controlled daily for detection of clinical symptoms ofadverse effects. For monitoring throughout the experiment, the bodyweight of the animals was documented two times weekly.

Acquisition of NIR Fluorescence Images

Non-invasive measurements or determination of NIR fluorescence signalscan be accomplished by labeling monoclonal antibodies with appropriateNIR fluorescence labels. E.g. different monoclonal antibodies werelabeled with a Cy5 or Cy5.5 or Cy7 dyes to monitor acquire NIRfluorescence images antibodies after i.v. injection into solid tumorcarrying mice and for monitoring the development of said solid tumorsand expression of tumor antigens originally overexpressed in said tumorsduring the treatment with non-labeled antibodies specifically binding tosaid tumor antigen. NIR fluorescence measurements were performed afterapplication of antibodies at different time points thereafter using theBonSAI Imaging System from Siemens Medizintechnik, Germany. By summingup mean intensities of the pixels in the region of the solid tumor a NIRfluorescence signal can be determined which is specific for the solidtumor in dependency of e.g. the expression level of the tumor antigen,the antibody labeled with a certain NIR fluorescence image, theacquisition time, the time after application of the labeled antibody andthe time after application of the non-labeled antibody intended for thetreatment of said solid tumor, etc.

Results

Example 1: NIR Fluorescence Imaging of Different Solid TumorsOverexpressing and not Overexpressing HER2 Tumor Antigen

Female SCID beige mice carrying BT474 or A549 tumors were injected withi.v. with a single dose of 20 μg/mouse of pertuzumab at a time pointwhen the tumor size was approximately 500 mm3. Tumor free SCID beigemice were also injected with the labeled monoclonal antibody and servedas a control. One day thereafter NIR fluorescence signal was measuredusing the BONSAI system (Siemens Medizintechnik). Acquisition time wasalways 2 seconds. Results depicted in FIG. 1a indicate that themonoclonal antibody pertuzumab labeled with Cy5 allows the detection ofHer2 overexpressing tumor cells BT474. In contrast, with A549 cells(FIG. 1b ) which do not express the Her2 antigen, a NIR fluorescencesignal was not detectable and also no NIR fluorescence signal could begenerated in tumor free mice (FIG. 1c ).

Example 2: NIR Fluorescence Imaging of Solid Tumors Overexpressing HER2Tumor Antigen

Female SCID beige mice carrying KPL-4 tumors were injected with Cy5labeled trastuzumab (FIG. 2a ) or Cy5 labeled pertuzumab (FIG. 2b ).Labeled antibodies were injected i.v. with a dose of 50 μg/mouse. Oneday thereafter NIR fluorescence signal was measured with an acquisitiontime of 4 seconds. The results (FIG. 2) indicate that using Cy5 labeledtrastuzumab (FIG. 2a ) or Cy5 labeled pertuzumab (FIG. 2b ) a comparableNIR fluorescence signal was detectable in the Her2 overexpressing cellline KPL-4.

Example 3 NIR Fluorescence Imaging of Solid Tumors Overexpressing HER2Tumor Antigen During the Treatment of a Patient with Non-LabeledTrastuzumab (Early Phase-48 h after First Trastuzumab Application)—Useof Pertuzumab Labeled with Cy5 (No Cross Reactivity with Trastuzumab)for NIR Fluorescence Imaging

Female SCID beige mice carrying KPL-4 tumors were injected i.p. with asingle dose of 30 mg/kg trastuzumab. Two days thereafter one group ofmice received Cy5 labeled trastuzumab (FIG. 3a ) and mice of the secondgroup were injected with Cy5 labeled pertuzumab (FIG. 3b ). There was nosignificant difference in the tumor size in the two groups and thelabeled antibodies were given i.v. at a dosage of 50 μg/mouse. NIRfluorescence signal was measured 24 hours thereafter with an acquisitiontime of 4 seconds. The background signal was 500 MFI (mean NIRfluorescence (NIRF) signal intensity [arbitrary units]) was measuredwith an acquisition time of 4 seconds. NIR fluorescence intensity wasquantified by summing up the number and signal intensities of the pixelsin the region of interest (ROI) or less were The results demonstratethat pre-injection with non-labeled trastuzumab prevents subsequentsignificant detection of Her2 expressing tumor cells when Cy5 labeledtrastuzumab is applied as a signal of 530 MFI in the region of the tumorwas measured, which is only little above the background signal of 500MFI (FIG. 3a ) Thus no significant NIR fluorescence signal can bedetected after a previous treatment with trastuzumab, so that Cy5labeled trastuzumab is not suitable for therapy monitoring during thetreatment of a patient with trastuzumab. In contrast, application of Cy5labeled pertuzumab (which binds to another epitope and is notcrossreactive with trastuzumab) gave a signal of 1440 MFI in the regionof the tumor (FIG. 3b ). Thus a significant NIR fluorescence signalcould be detected after a previous treatment with trastuzumab, so thatCy5 labeled pertuzumab is suitable for therapy monitoring during thetreatment with trastuzumab, as it shows a clearly improvedsignal/background ratio of 2.88 (=1440MFI/500MFI Cy5 labeled pertuzumabafter trastuzumab treatment) compared to the signal/background ratio of1.06 (=530MFI/500MFI—Cy5 labeled trastuzumab after trastuzumabtreatment). This allows a clearly better localization of the region oftumor than with the use of cross reactive antibodies; even when lessNIRF labeled antibody is given. The results also clearly show thatpertuzumab binds to an epitope which differs from the epitope recognizedby trastuzumab, and thus that trastuzumab and pertuzumab exhibit nocross reactivity against the same HER2 tumor antigen (while trastuzumaband Cy5 labeled trastuzumab exhibit cross reactivity and no significantNIR fluorescence signal can be detected after a previous treatment withtrastuzumab, so that Cy5 labeled trastuzumab is not suitable for therapymonitoring during the treatment with trastuzumab).

Example 4 NIR Fluorescence Imaging of Solid Tumors Overexpressing HER2Tumor Antigen During the Weekly Treatment with Non-Labeled Pertuzumab(48 h After Second Pertuzumab Application)—Use of Trastuzumab Labeledwith Cy5 (No Cross Reactivity with Pertuzumab) for NIR FluorescenceImaging

Female SCID beige mice carrying KPL-4 tumors were treated withpertuzumab injected i.p. twice weekly. The first injection (loadingdose) was 30 mg/kg and the second application (maintenance dose) was 15mg/kg (FIG. 4a ). Control animals received PBS only (FIG. 4b ). After 48hours mice of both groups were injected with 50 μg/mouse Cy5 labeledtrastuzumab and 4 days later NIR fluorescence signal was measured usinga acquisition time of 3 seconds. The figure FIG. 4a indicate that theNIRF signal in the pertuzumab treated mice is reduced compared to theNIR fluorescence signal in the PBS treated mice (FIG. 4b ). Thus, at thesolid HER2overexpressing KPL-4 tumor a response to the treatment withpertuzumab specifically binding to HER2 is detectable.

Example 5 NIR Fluorescence Imaging of Solid Tumors Expressing EGFR TumorAntigen During the Treatment of a Patient with Non-Labeled Cetuximab—Useof rhMab ICR62 Labeled with Cy5 (No Cross Reactivity with Cetuximab) forNIR Fluorescence Imaging

Female Balb/c nude mice carrying Calu3 tumors were injected i.p. withonce weekly for five weeks at a dose of 2.5 mg/kg Cetuximab. Two daysafter the last treatment mice received 2 mg/kg Cy5 labeled rhMab ICR62i.v. (FIG. 5). NIR fluorescence signal was measured 24 hours thereafterwith an acquisition time of 4 seconds. The results demonstrate thatpre-injection with non-labeled cetuximab allows subsequent detection ofEGFR expressing tumor cells and exact localization of the tumor tissuewhen Cy5 labeled rhMab ICR62 is applied (FIG. 5). This indicates thatrhMab ICR62 binds to an epitope which differs from the epitoperecognized by cetuximab, and shows that cetuximab and rhICR62 exhibit nocross reactivity against the same EGFR tumor antigen.

The invention claimed is:
 1. A method for near-infrared (NIR)fluorescence imaging of solid tumors in a patient during treatment witha first non-labeled monoclonal antibody, wherein the method comprises:a) administering to the patient a dose of the first non-labeledmonoclonal antibody, wherein the first non-labeled monoclonal antibodyspecifically binds to a tumor antigen in the solid tumor; b)administering to the patient a dose of a second monoclonal antibodylabeled with a NIR fluorescence label, wherein the second labeledmonoclonal antibody also specifically binds to the tumor antigen,wherein the first and second monoclonal antibodies exhibit no crossreactivity; and c) measuring the NIR fluorescence signal of the secondlabeled monoclonal antibody in a region of the solid tumor.
 2. Themethod of claim 1, wherein the signal/background ratio of the NIRfluorescence signal is at least 1.5.
 3. The method of claim 1, whereinthe first non-labeled monoclonal antibody is an anti-HER2 antibody. 4.The method of claim 3, wherein the first non-labeled monoclonal antibodyis trastuzumab or pertuzumab.
 5. The method of claim 1, wherein thefirst non-labeled monoclonal antibody is an anti-EGFR antibody.
 6. Themethod of claim 5, wherein the first non-labeled monoclonal antibody iscetuximab or rhMab ICR62.
 7. The method of claim 1, wherein the firstnon-labeled monoclonal antibody and the second labeled monoclonalantibody are co-administered to the patient during the treatment.
 8. Themethod of claim 7, wherein the first non-labeled monoclonal antibody andthe second labeled monoclonal antibody are co-administeredsimultaneously during the treatment.
 9. The method of claim 7, whereinthe first non-labeled monoclonal antibody and the second labeledmonoclonal antibody are co-administered sequentially in either orderduring the treatment.
 10. The method of claim 9, wherein the firstnon-labeled monoclonal antibody and the second labeled monoclonalantibody are co-administered in separate formulations.
 11. A method fortreating a patient suffering from a solid tumor overexpressing a tumorantigen, the method comprising: a) administering to the patient a firstdose of a monoclonal antibody labeled with a near-infrared (NIR)fluorescence label, wherein the labeled monoclonal antibody specificallybinds to the tumor antigen; b) measuring the NIR fluorescence signal ina region of the solid tumor, following the first dose of the labeledmonoclonal antibody; c) administering to the patient a first dose ofnon-labeled monoclonal antibody, wherein the non-labeled monoclonalantibody also specifically binds to the tumor antigen, wherein the firstand second monoclonal antibodies exhibit no cross reactivity; d)administering to the patient a second dose of the labeled monoclonalantibody labeled with a NIR fluorescence; e) measuring the NIRfluorescence signal in a region of the solid tumor, following the seconddose of the labeled monoclonal antibody; and f) administering to thepatient a second dose of the non-labeled monoclonal antibody, based on adecrease by at least 10% in the NIR fluorescence signal measured in stepe) compared to the NIR fluorescence signal measured in step b).
 12. Themethod of claim 11, wherein the administering in step f) is based on adecrease by at least 20% in the NIR fluorescence signal measured in stepe) compared to the NIR fluorescence signal measured in step b).
 13. Themethod of claim 11, wherein the administering in step f) is based on adecrease by at least 30% in the NIR fluorescence signal measured in stepe) compared to the NIR fluorescence signal measured in step b).
 14. Themethod of claim 11, wherein the non-labeled monoclonal antibody is ananti-HER2 antibody.
 15. The method of claim 14, wherein the non-labeledmonoclonal antibody is trastuzumab or pertuzumab.
 16. The method ofclaim 11, wherein the non-labeled monoclonal antibody is an anti-EGFRantibody.
 17. The method of claim 16, wherein the non-labeled monoclonalantibody is cetuximab or rhMab ICR62.
 18. A method for near-infrared(NIR) fluorescence imaging of solid tumors in a patient during treatmentwith a first non-labeled monoclonal antibody, wherein the methodcomprises: a) co-administering to the patient in a single formulation adose of the first non-labeled monoclonal antibody and a secondmonoclonal antibody labeled with a NIR fluorescence label, wherein thefirst non-labeled monoclonal antibody specifically binds to a tumorantigen in the solid tumor and the second labeled monoclonal antibodyalso specifically binds to the tumor antigen, wherein the first andsecond monoclonal antibodies exhibit no cross reactivity; and b)measuring the NIR fluorescence signal of the second labeled monoclonalantibody in a region of the solid tumor.
 19. The method of claim 18,wherein the signal/background ratio of the NIR fluorescence signal is atleast 1.5.
 20. The method of claim 18, wherein the first non-labeledmonoclonal antibody is an anti-HER2 antibody.
 21. The method of claim20, wherein the first non-labeled monoclonal antibody is trastuzumab orpertuzumab.
 22. The method of claim 18, wherein the first non-labeledmonoclonal antibody is an anti-EGFR antibody.
 23. The method of claim22, wherein the first non-labeled monoclonal antibody is cetuximab orrhMab ICR62.